The project will continue to be directed toward obtaining an understanding of the mechanism by which cellular Na+and K+ transport is regulated by thyroid hormone. In particular, the applicant will pursue the hypothesis proposed in the initial application for this Award that the well-known stimulation of active Na, K transport by thyroid hormone, rather than being attributable to a primary induction of the Na, K pump (Na, K- ATPase) as has previously been postulated, is instead due to an increase in the rate-limiting passive cellular influx of Na+ and efflux of K+. During the current project period the applicant has succeeded in obtaining substantial evidence for this hypothesis, including the first description of a continuous cell line (ARL 15 rat liver cells) which responds to thyroid hormone with an increase in active Na, K transport, and the further demonstration that this stimulation is indeed accompanied by corresponding increases in both passive Na+ influx and passive K+ efflux, as postulated. The availability of this convenient thyroid-hormone responsive cell culture system, and in particular of this convenient thyroid-hormone responsive cell culture system, and in particular of a second line of rat liver cells (Clone 9) which the applicant has more recently demonstrated to be similarly responsive to thyroid hormone, now make possible experiments designed to elucidate the specific mechanisms by which thyroid hormone regulates Na+ and K+ transport. The studies proposed for the requested 2-year project extension period will be carried out in the above mentioned Clone 9 cell culture system. The hypothesis that the effect of T3+ on active Na, K transport is attributable to an enhancement of the passive leak of Na and K, rather than to a primary induction of the Na, K- ATPase, will be directly tested by studying the time-course and concentration-response relationship of the effects of T3 on active Na, K transport, passive Na+ and K+ fluxes, intracellular Na+ and K+ concentrations, and Na, K-ATPase levels. Additionally, studies to characterize the specific membrane transport pathway(s) responsible for the T3-induced increase in passive Na+ entry and K+ efflux will be carried out using specific inhibitors and manipulations of the constituents of the medium. Finally, the possible role of changes in intracellular free calcium levels in mediating the effects of thyroid hormone will be investigated using fluorescence spectroscopy with calcium-sensitive dyes.